A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases.

Adenosine deaminases acting on RNA (ADARs) are endogenous enzymes catalyzing the deamination of adenosines to inosines, which are then read as guanosines during translation. This ability to recode makes ADAR an attractive therapeutic tool to edit genetic mutations and reprogram genetic information at the mRNA level. Using the endogenous ADARs and guiding them to a selected target has promising therapeutic potential. Indeed, different studies have reported several site-directed RNA-editing approaches for making targeted base changes in RNA molecules. The basic strategy has been to use guide RNAs (gRNAs) that hybridize and form a double-stranded RNA (dsRNA) structure with the desired RNA target because of ADAR activity in regions of dsRNA formation. Here we report on a novel pipeline for identifying disease-causing variants as candidates for RNA editing, using a yeast-based screening system to select efficient gRNAs for editing of nonsense mutations, and test them in a human cell line reporter system. We have used this pipeline to modify the sequence of transcripts carrying nonsense mutations that cause inherited retinal diseases in the FAM161A, KIZ, TRPM1, and USH2A genes. Our approach can serve as a basis for gene therapy intervention in knockin mouse models and ultimately in human patients.

The cycling and aging mouse female reproductive tract at single-cell resolution.

The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.

Recovery after inadvertent intramedullary microchip implantation at C1-C2 in a kitten.

Case summary: A 15-week-old male British Shorthair cat was presented for peracute paralysis immediately after microchip implantation. Neurological examination revealed a non-ambulatory tetraparesis and left thoracic limb plegia localised to C1-C5 spinal cord segments. CT of the cervical spine showed a diagonally orientated metallic foreign body (microchip transponder, 10 mm in length) within the vertebral canal at the level of C1-C2, resulting in a penetrating spinal cord injury. Based on concerns about further iatrogenic spinal cord injury through surgery, medical management was chosen. Despite the severe clinical signs, the kitten returned to ambulation within 6 days of the injury, with controlled urination and defecation. Continuous neurological improvement was seen for up to 6 weeks after the injury at which point a mild-to-moderate ambulatory tetraparesis and ataxia remained, with an overall good quality of life. Follow-up CT at the age of 13 months revealed a relative cranial displacement and rotation of the microchip towards the foramen magnum, while the cat's neurological status was unchanged. Relevance and novel information: This case demonstrated a cervical penetrating spinal cord injury in a growing cat caused by a microchip, which was successfully managed with medical treatment, suggesting that this might be an option for patients at risk of severe surgery-related complications or where owners reject surgery.

Inherited retinal diseases: Linking genes, disease-causing variants, and relevant therapeutic modalities.

Inherited retinal diseases (IRDs) are a clinically complex and heterogenous group of visual impairment phenotypes caused by pathogenic variants in at least 277 nuclear and mitochondrial genes, affecting different retinal regions, and depleting the vision of affected individuals. Genes that cause IRDs when mutated are unique by possessing differing genotype-phenotype correlations, varying inheritance patterns, hypomorphic alleles, and modifier genes thus complicating genetic interpretation. Next-generation sequencing has greatly advanced the identification of novel IRD-related genes and pathogenic variants in the last decade. For this review, we performed an in-depth literature search which allowed for compilation of the Global Retinal Inherited Disease (GRID) dataset containing 4,798 discrete variants and 17,299 alleles published in 31 papers, showing a wide range of frequencies and complexities among the 194 genes reported in GRID, with 65% of pathogenic variants being unique to a single individual. A better understanding of IRD-related gene distribution, gene complexity, and variant types allow for improved genetic testing and therapies. Current genetic therapeutic methods are also quite diverse and rely on variant identification, and range from whole gene replacement to single nucleotide editing at the DNA or RNA levels. IRDs and their suitable therapies thus require a range of effective disease modelling in human cells, granting insight into disease mechanisms and testing of possible treatments. This review summarizes genetic and therapeutic modalities of IRDs, provides new analyses of IRD-related genes (GRID and complexity scores), and provides information to match genetic-based therapies such as gene-specific and variant-specific therapies to the appropriate individuals.

Intracranial Subdural Fluid Accumulation Associated with a Choroid Plexus Carcinoma in a Dog.

Choroid plexus tumors are commonly described as intraventricular mass lesions and account for 7-10% of intracranial, primary tumors in dogs. A 3-year-old Shetland sheepdog was presented with a history of slowly progressive lethargy, vision impairment and cognitive deficits. On magnetic resonance imaging, a subdural fluid accumulation (SFA) overlying and compressing the left parietotemporal lobe as well as multifocal changes consisting of cyst-like lesions, supposed intra-axial brain lesions and mild, multifocal meningeal thickening and generalized contrast enhancement were identified. Cerebrospinal fluid (CSF) analysis showed a mononuclear pleocytosis with negative results for infectious agents. The dog was treated with prednisolone followed by burr hole craniotomy with puncture of the SFA, which macroscopically appeared to be CSF-like fluid. After initial improvement, the dog deteriorated despite continuation of prednisolone and cytarabine therapy and was euthanized four weeks after surgery. Histopathology was consistent with a disseminated, neuroinvasive choroid plexus carcinoma (CPC) that involved the entire neuroaxis including the meninges of the brain and spinal cord. Immunohistochemical examination showed a strong Kir7.1 and a heterogenous cytokeratin-immunoreactivity in neoplastic cells. In conclusion, a CPC should be considered as a possible cause of a SFA even in the absence of an intraventricular mass lesion.

Genetic counseling practice for inherited eye diseases in an Israeli medical center during the COVID-19 pandemic.

Inherited eye diseases (IED) are among the most common causes for childhood and young adulthood blindness in developed countries. Genetic counseling and testing have become an essential part of caregiving for families affected by one of these severe ocular pathologies. The objective of our study is to describe our experience during the 2020 (COVID-19) pandemic, following a practice protocol of safe genetic counseling for inherited ophthalmic diseases. We conducted a review of the genetic counseling practices from January until December 2020 in a multidisciplinary clinic for patients with visual impairment, in a tertiary hospital. The new protocol covered patient screening, required personal protective equipment, and the implementation of telemedicine. One hundred and eighty-three counseling sessions were done in this period of time; 33/183 were telemedicine counseling. The results of this study indicate that the practice of genetic counseling in regard to inherited eye diseases in the era of COVID-19 is effective and safe. Despite the high risk of infectivity that threatened healthcare professionals, safety measures adopted to reduce the risk of infection allowed us to prevent the cancelation of routine counseling, while keeping patient care our priority. The use of telemedicine was a very useful tool for providing counseling during lockdown periods in 2020.

SENIOR-LØKEN SYNDROME: A Case Series and Review of the Renoretinal Phenotype and Advances of Molecular Diagnosis.

PURPOSE: To report genetic and clinical findings in a case series of 10 patients from eight unrelated families diagnosed with Senior-Løken syndrome. METHODS: A retrospective study of patients with Senior-Løken syndrome. Data collected included clinical findings electroretinography and ocular imaging. Genetic analysis was based on molecular inversion probes, whole-exome sequencing (WES), and Sanger sequencing. RESULTS: All patients who underwent electrophysiology (8/10) had widespread photoreceptor degeneration. Genetic analysis revealed two mutations in NPHP1, two mutations in NPHP4, and two mutations in IQCB1 (NPHP5). Five of the six mutations identified in the current study were found in a single family each in our cohort. The IQCB1-p.R461* mutation has been identified in 3 families. Patients harboring mutations in IQCB1 were diagnosed with Leber congenital amaurosis, while patients with NPHP4 and NPHP1 mutations showed early and sector retinitis pigmentosa, respectively. Full-field electroretinography was extinct for 6 of 10 patients, moderately decreased for two, and unavailable for another 2 subjects. Renal involvement was evident in 7/10 patients at the time of diagnosis. Kidney function was normal (based on serum creatinine) in patients younger than 10 years. Mutations in IQCB1 were associated with high hypermetropia, whereas mutations in NPHP4 were associated with high myopia. CONCLUSION: Patients presenting with infantile inherited retinal degeneration are not universally screened for renal dysfunction. Modern genetic tests can provide molecular diagnosis at an early age and therefore facilitate early diagnosis of renal disease with recommended periodic screening beyond childhood and family planning.

Imepitoin for treatment of idiopathic head tremor syndrome in dogs: A randomized, blinded, placebo-controlled study.

BACKGROUND: Idiopathic head tremor syndrome is a paroxysmal movement disorder of unknown etiology. Spontaneous remission may occur, but owners may request treatment in severely affected dogs with continued episodes. Controlled studies of the disease are not available. HYPOTHESIS/OBJECTIVES: A drug with gamma amino butyric acid-ergic and anxiolytic effects will decrease head tremor episodes. ANIMALS: Twenty-four dogs with severe nonremitting head tremor and presumptive clinical diagnosis of idiopathic head tremor syndrome. METHODS: Prospective, blinded, placebo-controlled clinical trial to compare imepitoin with placebo in dogs with frequent episodes of idiopathic head tremor. Evaluation of efficacy used the quotient T2/T1 that represented prolongation of the head tremor-free period compared to a 3-month baseline. A dog was considered a responder if tremors subsided or if the head tremor-free period was 3× longer than the longest period during baseline (T2/T1 ≥ 3). Sample size calculations considered a large effect of imepitoin on T2/T1 (Cohen's d = 0.8). RESULTS: There were no responders in the placebo group (0/12). In the imepitoin group, the responder rate was 17% (2/12; P = .18) with T2/T1 3.8 and 4.0. Mean T2/T1 was 1.0 ± 1.4 in the imepitoin and 0.4 ± 0.4 in the placebo group (P = .37). CONCLUSION AND CLINICAL IMPORTANCE: Imepitoin did not result in a significant overall benefit. Future studies should focus on treatment of subgroups with a common pathophysiology and similar comorbidities.

Synthesis of the Anionic Hydroxypropyl-ß-cyclodextrin:Poly(decamethylenephosphate) Polyrotaxane and Evaluation of its Cholesterol Efflux Potential in Niemann-Pick C1 Cells.

Niemann-Pick type C disease (NPC) is a lysosomal storage disease that is characterized by a progressive accumulation of unesterified cholesterol in the lysosomes leading to organ damage from cell dysfunction. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is an attractive drug candidate for treating NPC, as it diminishes cholesterol accumulation in NPC cells. Systemic HP-ß-CD treatment, however, is limited by rapid renal clearance. We designed a new anionic HP-ß-CD polyrotaxane to act as a slow release formulation based on a polyalkylene phosphate core to improve the pharmacokinetics. The polyalkylene phosphate comprises hydrophobic decamethylene spacers linked by biodegradable anionic phosphodiester bonds. HP-ß-CD was threaded onto this polymer first and α-CD afterwards to prevent burst release of the threaded HP-ß-CD. Our findings show that HP-ß-CD was slowly released from the watersoluble polyrotaxane over a 30 days period. The polyrotaxane provided persistently diminished cholesterol levels in NPC1 cells by 20% relative to untreated cells. These results demonstrate the therapeutic potential of this novel HP-ß-CD polyrotaxane for the mobilization of aberrantly stored cholesterol in NPC1 cells.

The Adequacy of Core Biopsy in the Assessment of Smooth Muscle Neoplasms of Soft Tissues: Implications for Treatment and Prognosis.

The grading of soft tissue sarcomas is one of the most important prognostic factors and determines patient management. Although grading of most adult-type soft tissue sarcomas on biopsies correlates highly with the final grading on the excision specimen, it appears less reliable for tumors of smooth muscle. We assessed the pathologic findings for smooth muscle neoplasms diagnosed by core biopsy at our tertiary sarcoma center, and compared them with those in the subsequent excision specimens. A total of 100 patients with leiomyosarcoma first diagnosed on core biopsy and with a subsequent excision were identified and the accuracy of the biopsy grade determined by comparison with the excision grade. Differences in other salient histologic parameters were also noted. A grade difference between biopsy and excision specimens of leiomyosarcomas was found in 68% of cases, with all these cases showing an increase in grade from biopsy to excision specimen. Of the 3 parameters used for grading using the French Federation of Cancer Centers Sarcoma Group Grading System (FNCLCC), necrosis was the score that most commonly differed between biopsy and excision specimen (55%), closely followed by the mitotic count (51%). The grading of soft tissue smooth muscle tumor biopsies has a lower accuracy compared with other adult soft tissue sarcomas and should therefore be taken with caution, particularly as this may be an underrepresentation of the true tumor grade.

Lay public mental models of ionizing radiation: representations and risk perception in four European countries.

How do members of the lay public understand ionizing radiation and its effects? How do they define ionizing radiation, and to which other concepts do they connect it? Do they perceive danger and risks? Do they know how to protect themselves? What do they think of various applications of ionizing radiation? Where and how does the public lodge confidence or doubt? What is the demand for information and transparency? We applied the mental models approach to investigate these questions in four European countries (France, Poland, Romania, and Slovenia; 63 respondents), providing a rich picture of how the lay public perceives ionizing radiation. Mental models are messy and overlapping but meaningful and useful schemas, resulting from a person's multidimensional experience and relation to the surrounding natural and social world. Collectively, members of the lay public (independently of their education or background) possess a non-negligible amount of knowledge on the topic of ionizing radiation risk, and hold strong views on related concepts. Mutual understanding between specialists and potentially affected citizens may be a critical pathway to build a shared and effective risk culture supported by accurate, two-way risk communication and inter-actor relationships.

Well-Differentiated Liposarcoma With Hibernoma-Like Morphology.

Well-differentiated liposarcoma (WDL) can show a morphologic spectrum, including lipoma-like, sclerosing and inflammatory subtypes. It does not metastasize but can dedifferentiate, acquiring metastatic potential. Hibernomas are benign neoplasms that show variable differentiation toward brown fat, and can sometimes occur in the abdomen or retroperitoneum. We illustrate a case of retroperitoneal WDL that showed extensive hibernoma-like morphology, with sheets of multivacuolated adipocytes of varying sizes, with abundant cytoplasm and numerous lipid vacuoles or granular eosinophilic cytoplasm. However, very focally there were fibrous septa containing spindle cells with enlarged, hyperchromatic, mildly pleomorphic nuclei. Further sampling showed areas of typical WDL, with lobules of mature fat intersected by fibrous septa containing atypical, enlarged spindle cells, as well as small foci of dedifferentiation. Immunohistochemistry for CDK4 and p16 showed strong and diffuse nuclear expression in the hibernoma-like areas, and fluorescence in situ hybridization showed MDM2 gene amplification, all in keeping with WDL. We highlight hibernoma-like morphology as part of the histologic spectrum of WDL, and recognition of this variant is important for correct treatment and prognostication.

Ossifying fibromyxoid tumor: morphology, genetics, and differential diagnosis.

Ossifying fibromyxoid tumor (OFMT) is a soft tissue neoplasm of uncertain differentiation and intermediate (rarely metastasizing) biologic potential, with typical morphologic features, of an encapsulated, lobulated tumor comprising uniform polygonal cells within fibromyxoid stroma, which is surrounded by or contains metaplastic bone, classically as a peripheral rim of lamellar bone. Ossifying fibromyxoid tumor can arise at almost any site, although most frequently occurs within the extremities and trunk. Although most behave in a benign fashion, tumors can rarely show atypical or malignant features. It is now established that OFMTs represent translocation-associated tumors, with up to 85% associated with recurrent gene rearrangements, mostly involving the PHF1 gene (including in typical, atypical, and malignant neoplasms), with EP400-PHF1 in approximately 40% of tumors, and ZC3H7B-BCOR, MEAF6-PHF1, and EPC1-PHF1 fusions also described. Correct diagnosis is clinically important to ensure correct treatment and prognostication, both to avoid overdiagnosing OFMT as a malignant neoplasm such as osteosarcoma and also because of the propensity for aggressive behavior in a small number of OFMT. We review OFMT, with emphasis on the morphologic spectrum, recent molecular genetic findings, and the differential diagnosis.

NOD2 mutations are associated with the development of intestinal failure in the absence of Crohn's disease.

BACKGROUND & AIMS: Short bowel syndrome (SBS) and intestinal failure (IF) are multi-factorial conditions which in adults result from extensive intestinal resection. NOD2 is an intracellular pattern recognition receptor associated with CD. An unexpected high frequency of NOD2 mutations has been found in patients undergoing intestinal transplantation (35%). The role of NOD2 in a cohort with SBS/IF not specifically requiring intestinal transplantation has not been studied yet. METHODS: The course of 85 patients with non-malignant SBS/IF was characterized. The major NOD2 mutations, as well as ATG16L1 and IL23R were determined. The allele frequencies were compared to the published frequencies of CD patients and controls. RESULTS: In non-CD patients (72%) allele frequencies of NOD2 mutations were statistically more frequent than in controls (14% vs 6%, p = 0.006). In CD patients (28%) allele frequencies were not different between SBS and controls (29% vs 22%, p = 0.23). NOD2 mutations were neither associated with parameters potentially heralding the need for transplantation nor with an earlier time to the indication for intestinal transplantation. CONCLUSIONS: NOD2 mutations are associated with the development of SBS/IF in the absence of CD, but not with specific complications. NOD2 mutations may increase the risk for more extensive intestinal resection or may impair intestinal adaptation.

An internet survey of use, opinions and preferences for smoking cessation medications: nicotine, varenicline, and bupropion.

INTRODUCTION: We assessed use, compliance and preferences among smoking cessation medications in a "real world" sample of current and former smokers. METHODS: Internet survey on a smoking cessation website (French/English, 2008-2010) to assess use of nicotine replacement therapies (NRT), varenicline, and bupropion. RESULTS: There were 885 participants (39% current smokers, 61% former smokers), the majority of the sample (70%) was female. The most frequently used medications were, in order: patches (40%), varenicline (23%), nicotine gum (16%), nicotine lozenge/tablet (10%), bupropion (8%), and inhaler (3%). Satisfaction, perceived relief of craving/withdrawal and effectiveness were best for varenicline and lowest for gum. In current users, duration of use was longest for gum (121 days), lozenge/tablet (152 days) and shortest for patch (25 days). Daily use was good for lozenge (9 pieces/day) but less than recommended for gum (6 pieces/day) and inhaler (2 plugs/day). People who tried more than 1 medication found varenicline more effective and satisfactory than NRT or bupropion; and users preferred patch to gum. By smoking status, former smokers had more education, reported greater use of medications (daily, over time) and reported more satisfaction with medications than current smokers. CONCLUSIONS: An Internet survey showed smoking cessation medications differed significantly in perceived effectiveness, satisfaction, and smoking status (former vs. current smoking). Except for lozenge/tablet, insufficient daily use remained a problem with acute NRTs. For all medications, improving outcome may require better instruction for proper use, approval of new indications (precessation) or development of new medications that bypass compliance issues that undermine success.

Comparison of craving and withdrawal among four combination nicotine treatments.

OBJECTIVE: To assess the appearance of craving and withdrawal among four combination nicotine replacement treatments (NRTs). METHODS: In a crossover trial of NRT preferences, 27 smokers tested 4 combinations of nicotine treatments: 2 mg/4 mg gums + 15 mg patch, 2 mg/4 mg lozenges + 15 mg patch, inhalers + 15 mg patch, and 10 + 15 mg double patch (approximately 25 mg). Overnight abstinence was required prior to (1/2) day testing of each combination. Combination NRTs were used for approximately 6 h/day. Subjects resumed smoking each afternoon. For this report, we used the Smoker Anchored Withdrawal Grid to look at craving and withdrawal scores over 5 days of testing (smoking baseline + four treatment days). RESULTS: "Urge to smoke" and "total withdrawal" showed a rise from baseline to NRT use for the double patch but not for the three acute + patch conditions. Lozenge/patch scores did not rise from baseline for "craving" and "miss a cigarette" but did for gum/patch, inhaler/patch, and double patch. The best relief occurred for NRTs of choice. CONCLUSION: This was a small but suggestive finding regarding the potential of patch plus adjunct ad lib NRT. With little data on relief with NRT combinations, more systematic tests are needed.

Preferences among five nicotine treatments based on information versus sampling.

Smokers' minimal exposure to nicotine replacement treatments (NRTs) may account for poor compliance and outcome with these treatments. This study tested effects of information versus sampling of NRTs on smokers' preferences and expectations. The study was a crossover comparing information-only (INF) with sampling (SMP) methods for five NRTs: gum (2 and 4 mg), lozenges (2 and 4 mg), and inhalers. Subjects were given computer-based presentations on NRTs (INF) and rated and ranked use variables (e.g., ease, sensory/ritual, perceived relief, embarrassment) and overall choice for "use to quit." After INF testing, subjects sampled each NRT (SMP) and again rated and ranked drugs. SMP was brief (4 min) to mimic potential use in practice. Results showed changes in perceptions and preferences post-SMP. NRT preferences shifted for overall "use to quit" (59%) and most use variables (43%-63%) post-SMP. Inhalers (generally top choice) showed a 20% drop in choice to quit (p<.04) and a 24% drop in anticipated "relief of withdrawal" (p<.04) post-SMP; 4-mg lozenge ratings increased for "relief of withdrawal" (p<.02). Ratings improved post-SMP for three of the five NRTs ("ease of use," p<.05) but were reduced overall for liking "sensory action" (p<.003) and reduced for all but 2-mg gum for "use to quit" (p<.03). Positive changes were seen in improved ratings of NRTs chosen post-SMP. Given that reactions to NRTs change with experience, sampling should allow for a more realistic choice of NRT (self-tailoring) and better compliance versus current trial-and-error methods.

Preferences among four combination nicotine treatments.

RATIONALE: Acute nicotine replacement treatments (NRTs) are disliked or misused, leading to insufficient nicotine intake and poor outcome. Patches provide steady nicotine but are slow and passive. Combining systems may improve efficacy with acute NRTs tailored for compliance. OBJECTIVE: To test initial reactions to and use preferences among combinations of NRTs. MATERIALS AND METHODS: Smokers (n=27) tested four combination NRTs in a 5-day crossover trial: 2/4-mg gum + 15-mg patch (G/P), 2/4-mg lozenges + 15-mg patch (L/P), inhaler + 15-mg patch (I/P), and 10 mg + 15-mg patches (P/P). Subjects rated an NRT combination each day after 5-6 h of use and ranked among the NRTs after testing all treatments. RESULTS: Double-patches (P/P) were ranked highest for "ease of use", "safety", and "use in public". However, for "help to quit", 70% preferred some form of acute-patch combination (A/P) compared to 30% choosing P/P. For "use under stress" (an immediate need), 93% preferred A/P systems compared to 7% choosing P/P. L/P ranked lowest for "ease of use", I/P and L/P were lowest on "safety", and I/P ranked lowest for "use in public". Expectations of NRTs changed with test experience for patches (better) and lozenges (worse). CONCLUSIONS: In brief testing, all combinations were acceptable. P/P was favored for ease, safety, and public use, but a majority chose A/P systems for help in quitting and use under stress. Combined use is viable and needs to be made known and accessible to smokers.

Comparison of three nicotine treatments: initial reactions and preferences with guided use.

RATIONALE: Misuse or dislike of nicotine replacement treatments (NRTs) undermines their effectiveness. Brief testing among NRTs could allow tailoring by preference to improve outcome. OBJECTIVE: To test initial reactions/preferences to NRTs in a single session crossover design with guided use. METHODS: Smokers were offered two doses of three NRTs: gum (2 and 4 mg), inhaler, and nasal spray (NNS) in a 5-h test with proper use enforced. Subjects rated each NRT and ranked among NRTs on use variables and preferences. RESULTS: Gum was ranked over inhaler and NNS for "ease of use," "safety" and "prefer in public." Four-milligram gum was rated higher than 2 mg on several variables. With experience, "ease of use" and "liking" improved for gum. Both inhaler and NNS ranked low on considering "use >3 months" vs gum. Dislike of NRT was reflected in refusal of second doses. For those testing all doses (n=9), inhaler ranked last on "relief of withdrawal," "choose under stress," and "choice to help quit." Craving and withdrawal were relieved over time with any NRT use. CONCLUSIONS: Sampling of treatments can identify reactions key to initial compliance with these NRTs.